Bicc1 and dicer regulate left-right patterning through post-transcriptional control of the Nodal-inhibitor dand5

Abstract

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. How the flow signal is processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal drives LPM Nodal cascade induction. Here, we identify the mechanism of dand5 downregulation, finding that its posttranscriptional repression is a central process in symmetry breaking. Specifically, the RNA binding protein Bicc1 interacts with a proximal element in the 3'-UTR of dand5 to repress translation in a dicer1-dependent manner. The bicc1/dicer1 module acts downstream of flow, as LRO ciliation was not affected upon its loss. Loss of bicc1 or dicer1 was rescued by parallel knockdown of dand5, placing both genes in the process of flow sensing.

Dual modulation of phase-transitioning licenses the Bicc1 network of ciliopathy proteins to bind specific target mRNAs

“…However, both our gainof-function studies in HEK293T cells and the regulation of endogenous Bicc1 by Anks3 knockdown in mIMCD3 cells support the conclusion of our cell-free assays that the threshold of mRNA binding is greatly increased by Anks3-mediated inhibition, reducing the background noise of non-specific binding and allowing ANKS6 to spatiotemporally couple the recruitment of specific target mRNAs to Bicc1 phase transitioning in metronomically regulated amounts. While Dand5 mRNA is also below detection in murine IMCD3 kidney cells, its 3'-UTR has been validated in vivo as a conserved target mediating left-right axis specification in response to cilia-induced activation of Bicc1 preferentially on the future left side (Maerker et al, 2021;Minegishi et al, 2021). Here, we observed increased binding of endogenous Bicc1 to its own mRNA and to endogenous Adcy6 transcripts upon RNAi depletion of ANKS3 in IMCD3 cells.…”

“…Consistent with a role in activating Ccr4-Not, Drosophila Bicc1 binds the Cnot3/5 subunit and promotes deadenylation of its own mRNA during the initial stages of oogenesis (Chicoine et al, 2007). In addition, loss-of-function studies revealed that Cnot3 and Bicc1 accelerate the decay of Dand5 mRNA to specify the left-right asymmetry of visceral organs in vertebrates (Maerker et al, 2021;Minegishi et al, 2021). Regulated Dand5 mRNA decay depends on the proximal 3'-UTR, which is recognized by the KH1 and KH2 domains of Bicc1 via a bipartite GAC motif in the conserved GACGUGAC sequence, and on flow stimulation of polycystin-2 in primary cilia on the left side of the node (Minegishi et al, 2021).…”

Quantitative analysis of transcriptome dynamics provides novel insights into developmental state transitions

“…This concentration of BMP4/7 was utilized as it induced levels of pSmad158 at stage 10 that were comparable to levels present at stage 10.5 in untreated caps. For morpholino experiments, a previously validated translation-blocking dand5 morpholino [116,127]. (Gene Tools, Sequence: 'CTG GTG GCC TGG AAC AAC AGC ATG T ') was injected in 4 cells at the eight-cell stage for a total of 40 pmol per embryo.…”

Getting to the Heart of Left–Right Asymmetry: Contributions from the Zebrafish Model

“…As well as work outlined in this review, there are several studies emerging in online repositories that provide hints of forthcoming areas of research in this field. These include the regulation of asymmetric peri-KV expression [73], control of lefty expression dynamics [74] and mapping of cell behaviours to analyse looping morphogenesis [75]. Whilst much headway has been made in the past two decades, there remains much unknown about the mechanisms of asymmetric heart formation, particularly with regard to the later stages of morphogenesis.…”